The proposed experiments are a continuation of our studies on the determinants of cardiac phenotype during normal development and in altered functional states of the heart. Since cAMP plays an important role in cardiac contractility, we will study its role in modulation of alpha-myosin heavy chain (MHC) expression. Our preliminary studies have shown that forskolin induces a several fold increase in the steady state level of MHC-alpha mRNA in serum-pretreated primary cultures derived from fetal rat hearts while no change was observed in the abundance of MHC-beta transcript. Therefore, we will focus primarily on the transcriptional regulation of the alpha-MHC gene. We will first complete our ongoing characterization of the cAMP response in cultured myocytes by comparing the steady level of mRNA with its transcription rate and by evaluating the mRNA stability. Next, to delineate the mechanism(s) of serum induced cAMP transcriptional regulation, we will study the promoter activity using transient transfection assays of primary myocyte cultures. Both the regulatory sequences and their cognate transcription factor(s) responsible for the serum induced cAMP effect will be investigated and their possible interaction evaluated. Thus, our specific aims are to: 1. Investigate the level of regulation of MHC-alpha gene expression by cAMP. a. Analyze the cAMP transcriptional regulation of MHC-alpha gene expression. b. Determine the effect of cAMP on MHC-alpha mRNA stability. 2. Study the promoter function in response to cAMP. a. Delineate the cAMP responsive element (AP-2E) and serum response element (SRE) within MHC-alpha promoter. b. Study the role of respective transcription factors, AP-2 and SRF on the MHC-alpha gene expression.